The present invention relates to the gene encoding an enzyme that exhibits both alkaline pullulanase activity and alkaline xcex1-amylase activity (alkaline amylopullulanase), to alkaline xcex1-amylase and alkaline pullulanase obtainable from expression of a fragment of the gene encoding the intact alkaline amylopullulanase, to the gene or fragments thereof encoding these enzymatic activities, and to recombinant DNAs and transformants bearing the gene and fragments thereof.
Alpha-amylase has long been used in a variety of fields. For example, it has been used for the saccharification of grains and potatoes in the fermentation industry, as starch paste removers in the textile industry, as digestives in the pharmaceutical industry, and for the manufacture of thick malt sugar syrups in the food industry. Alpha-amylase is an enzyme which acts on starch-related polysaccharides such as amylose or amylopectin, cutting solely the xcex1-1,4-glucoside bond of the polysaccharide molecule. Crystalline samples or electrophoretically uniform samples of xcex1-amylase have been obtained from a number of different sources including bacteria, fungi, plant seeds, and animal digestive glands. Pullulanase is an enzyme which hydrolyzes solely the xcex1-1,6-glucoside bond present in starch, glycogen, amylopectin, and pullulan. Pullulanase was first found in a certain strain of Aerobacter aerogenes (Bender, H. and Wallenfels, K., Biochem. J., 334, 79 (1961)), and thereafter, was also found in many other microorganisms including genera Bacillus, Streptococcus and Clostridium. Pullulanase has become of interest in the starch-making industry because of its ability to produce, from starch, maltooligosaccharides such as glucose, maltose, maltotriose, maltopentaose, and maltohexaose when it is used in combination with endo-type amylase and exo-type amylase.
In order to simplify the process of the manufacture of saccharides in which two or more enzymes are used, as described above, pullulanase which also acts on the xcex1-1,4-glucoside bond, in other words, pullulanase exhibiting xcex1-amylase activity, is greatly desired. Bacillus subtilis TU strain is known to produce a pullulanase-amylase complex enzyme (Takasaki, Y., Agric. Biol. Chem., 51, 9 (1987), Japanese Patent Publication (kokoku) No. 1-18717). In addition, enzymes exhibiting the above two distinct enzymatic activities or so-called amylopullulanases have been reported for a number of bacteria including Bacillus circulans (Japanese Patent Application Laid-open (kokai) No. 64-60376), Bacillus sp. (Saha, B. C., et al., Enzyme Microb. Technol., 11, 760 (1989)), Thermoanaerobium brockii (Coleman, R. D. et al., J. Bacteriol., 169, 4302 (1987)), Thermoanaerobium sp. (Plant, A. R., et al., Appl. Microbiol. Biotechnol., 26, 427 (1987)), Clostridium thermohydrosulfuricum (Saha, B. C., et al., Biochem. J., 252, 343 (1988)), Clostridium thermosulfurogenes (Spreinat, A. et al., Appl. Microbiol. Biotechnol., 33, 511 (1990)), Thermus aquaticus (Plant, A. R., et al., Enzyme Microb. Technol., 8, 668 (1986)), Thermus sp. (Nakamura, N et al., Starch/Starke, 41, 112 (1989)), Thermoanaerobacterium saccharolyticum (Saha, B. C., et al., Appl. Environ. Microbiol., 56, 881 (1990)), and Pyrococcus furiosus and Thermococcus litoralis (Brown, S. H. and Kelley, R. M., Appl. Environ. Microbiol., 59, 2614 (1993)).
The present inventors have recently discovered that the efficacy of dish-washing detergents and detergents for clothes can be greatly improved, particularly on starch soils, when xcex1-amylase and pullulanase are both incorporated into the detergents (Japanese Patent Application Laid-open (Kokai) No. 2-132193). However, most of the xcex1-amylases and pullulanases previously found in the natural world exhibit maximal and stable enzymatic activities in the neutral to acidic pH ranges, but scarcely work in an alkaline solution of pH 9-10. There exist very few enzymes exhibiting maximal activities in the alkaline pH range (alkaline pullulanases), and only two reports of such enzymes have been published (Nakamura, N. and Horikoshi, K., Biochim. Bophys. Acta, 397, 188 (1975), Japanese Patent Publication (kokoku) No. 53-27786 and Ara et al. Japanese Patent Publication kokoku) No. 6-32613. Furthermore, an enzyme that has both alkaline xcex1-amylase and alkaline pullulanase activities had not been reported until the present inventors discovered that an alkalophilic Bacillus sp. KSM-AP1378 (FERM BP-3048, deposited Jul. 24, 1989 in Fermentation Research Institute, Agency of Industrial Science and Technology of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, 305 Japan) having its optimum pH for growth in the alkaline range produces a novel alkaline amylopullulanase (formerly designated pullulanase Y) that has both alkaline pullulanase and alkaline xcex1-amylase activities. They elucidated that this enzyme is useful as an additive in detergent compositions for automatic dishwashers and in detergent compositions for clothes (Japanese Patent Application Laid-open (kokai) No. 3-290498). Although this enzyme is constituted by a single enzyme molecule, it exhibits both alkaline xcex1-amylase activity and alkaline pullulanase activity. Exploitation of this enzyme has proved very advantageous in culturing the bacteria and in purification of the enzyme, compared to the case in which the two enzymes are independently produced by two different bacteria.
The present inventors have attempted to improve productivity of the alkaline amylopullulanase (formerly designated pullulanase Y) producing bacterium, Bacillus sp. KSM-AP1378, through optimization of culturing methods. Nevertheless, it is still desired to improve further the enzyme productivity of the bacterium so as to advantageously produce the alkaline amylopullulanase on an industrial scale. It is noted that production of the enzyme can be further enhanced using genetic engineering and the activity of the enzyme itself can be improved by altering the gene encoding the enzyme using a protein engineering approach. Applying these approaches requires the gene encoding alkaline amylopullulanase.
Accordingly, an object of the present invention is to provide the gene encoding alkaline amylopullulanase, recombinant DNA comprising the gene, and a transformant harboring the recombinant DNA.
The DNA encoding the alkaline amylopullulanase gene may be further used to produce probes to be used in the isolation of additional, homologous alkaline amylopullulanase genes from other microorganisms. Thus, an additional object of the present invention is to provide a means of screening for and isolating additional alkaline amylopullulanase enzymes.
The present inventors isolated a DNA fragment encoding alkaline amylopullulanase from the chromosomal DNA of an alkalophilic Bacillus strain using shotgun cloning and PCR. When they transformed a microorganism with this DNA fragment ligated to a suitable vector, it was confirmed that the resultant recombinant microorganism produced alkaline amylopullulanase. Moreover, it was found that the amino acid sequence of the alkaline amylopullulanase encoded by the DNA fragment is completely different from those of previously known amylases and pullulanases, and that this enzyme has the feature that the amino terminal moiety of the enzyme molecule is alkaline xcex1-amylase, and the carboxy terminal moiety of the enzyme molecule is alkaline pullulanase. The present invention was accomplished based on this finding.
Accordingly, the present invention provides a DNA fragment encoding alkaline amylopullulanase.
The present invention also provides alkaline xcex1-amylase having the amino acid sequence described in SEQ ID NO:3 provided hereinbelow, as well as a DNA fragment encoding the alkaline xcex1-amylase.
The present invention also provides alkaline pullulanase having the amino acid sequence described in SEQ ID NO:4 provided hereinbelow, as well as a DNA fragment encoding the alkaline pullulanase.
The present invention also provides recombinant DNA comprising a DNA fragment encoding the above-described alkaline amylopullulanase, alkaline xcex1-amylase, or alkaline pullulanase.
The present invention also provides a transformed microorganism harboring recombinant DNA comprising a DNA fragment encoding the above-described alkaline amylopullulanase, alkaline xcex1-amylase, or alkaline pullulanase.
The present invention further provides a method for producing alkaline amylopullulanase, alkaline xcex1-amylase, or alkaline pullulanase, characterized by culturing the above-described transformed microorganism and collecting any one of the expressed enzymes.